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Construction of protein-responsive shRNA/RNAi control system using RNP motif

Foreign code F120006334
File No. I029P008WO
Posted date Mar 22, 2012
Country United States of America
Application number 200913133709
Gazette No. 20110263026
Gazette No. 8871437
Date of filing Dec 9, 2009
Gazette Date Oct 27, 2011
Gazette Date Oct 28, 2014
International application number JP2009070580
International publication number WO2010067811
Date of international filing Dec 9, 2009
Date of international publication Jun 17, 2010
Priority data
  • P2008-312951 (Dec 9, 2008) JP
  • 2009WO-JP70580 (Dec 9, 2009) WO
Title Construction of protein-responsive shRNA/RNAi control system using RNP motif
Abstract (US8871437)
An object of the present invention is to provide an RNAi control system using an RNA-protein interaction motif.
The present invention provides an shRNA comprising: a guide strand having a sequence complementary to a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, wherein the linker strand comprises an RNP-derived protein-binding motif sequence.
The present invention also provides an RNAi control system comprising: the shRNA; and an RNP-derived protein which specifically binds to a protein-binding motif sequence in the shRNA.
Scope of claims [claim1]
1. An RNAi control system responsive to a protein expressed in a cell, the system comprising: a vector for expression of an shRNA comprising: a guide strand having a sequence complementary to an mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence,
wherein the binding of an L7Ae protein or L7Ae protein-containing fusion protein expressed in the cell to the shRNA inhibits the cleavage of the shRNA by Dicer.
[claim2]
2. An RNAi control method responsive to a protein expressed in a cell, the method comprising the step of: introducing into the cell a vector for expression of an shRNA comprising a guide strand having a sequence complementary to an mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence,
wherein the binding of an L7Ae protein or L7Ae protein-containing fusion protein expressed in the cell to the shRNA inhibits the cleavage of the shRNA by Dicer.
[claim3]
3. The RNAi control system according to claim 1, wherein the target sequence of the shRNA is Bc1-xL mRNA, and the RNAi control system controls the expression of an apoptosis regulatory protein.
[claim4]
4. An shRNA comprising: a guide strand having a sequence complementary to an mRNA of a target sequence; a passenger strand which forms a duplex with the guide strand; and a linker strand which links the guide strand and the passenger strand, the linker strand comprising a Box CD sequence, wherein in response to an L7Ae protein or L7Ae protein-containing fusion protein expressed in the cell, the cleavage of the shRNA by Dicer is inhibited to control the expression of a protein encoded by the mRNA of the target sequence.
[claim5]
5. The RNAi control system according to claim 1, further comprising a vector for intracellular expression of the L7Ae protein or L7Ae protein-containing fusion protein.
[claim6]
6. The RNAi control system according to claim 1, wherein the target sequence of the shRNA is GFP mRNA.
[claim7]
7. The RNAi control method according to claim 2, wherein the target sequence of the shRNA is Bc1-xL mRNA, and the RNAi control method controls the expression of an apoptosis regulatory protein.
[claim8]
8. The RNAi control method according to claim 2, further comprising the step of introducing a vector for intracellular expression of the L7Ae protein or L7Ae protein-containing fusion protein into the cell.
[claim9]
9. A method for quantifying the expression of an intracellular marker protein without destroying a cell, comprising the steps of: introducing to the cell the shRNA of claim 4 wherein the target sequence is a GFP mRNA; and
measuring the fluorescence intensity of the GFP.
  • Inventor, and Inventor/Applicant
  • INOUE TAN
  • SAITO HIROHIDE
  • KASHIDA SHUNICHI
  • HAYASHI KARIN
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
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